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Original article
Genetic editing of colonic organoids provides a molecularly distinct and orthotopic preclinical model of serrated carcinogenesis
  1. Tamsin R M Lannagan1,
  2. Young K Lee1,
  3. Tongtong Wang1,
  4. Jatin Roper2,3,
  5. Mark L Bettington4,5,
  6. Lochlan Fennell5,6,
  7. Laura Vrbanac1,
  8. Lisa Jonavicius7,
  9. Roshini Somashekar1,
  10. Krystyna Gieniec1,
  11. Miao Yang1,
  12. Jia Q Ng1,
  13. Nobumi Suzuki1,
  14. Mari Ichinose1,
  15. Josephine A Wright1,
  16. Hiroki Kobayashi1,
  17. Tracey L Putoczki8,
  18. Yoku Hayakawa9,
  19. Simon J Leedham10,11,
  20. Helen E Abud12,
  21. Ömer H Yilmaz2,13,
  22. Julie Marker14,
  23. Sonja Klebe7,
  24. Pratyaksha Wirapati15,
  25. Siddhartha Mukherjee16,
  26. Sabine Tejpar17,
  27. Barbara A Leggett5,18,19,
  28. Vicki L J Whitehall5,19,20,
  29. Daniel L Worthley1,
  30. Susan L Woods1
  1. 1School of Medicine, University of Adelaide and South Australian Health and Medical Research Institute, Adelaide, South Australia, Australia
  2. 2The David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, Massachusetts, USA
  3. 3Division of Gastroenterology, Tufts Medical Center, Boston, Massachusetts, USA
  4. 4Envoi Specialist Pathologists, Brisbane, Queensland, Australia
  5. 5Conjoint Gastroenterology Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia
  6. 6School of Health and Sports Science, University of the Sunshine Coast, Brisbane, Queensland, Australia
  7. 7Department of Anatomical Pathology, Flinders Medical Centre, Bedford Park, South Australia, Australia
  8. 8Department of Medical Biology, University of Melbourne and the Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia
  9. 9Department of Gastroenterology, University of Tokyo, Tokyo, Japan
  10. 10Translational Gastroenterology Unit, Experimental Medicine Division, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford, UK
  11. 11Gastrointestinal Stem Cell Biology Laboratory, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK
  12. 12Cancer Program, Monash Biomedicine Discovery Institute and the Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia
  13. 13Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA
  14. 14Cancer Voices SA, Adelaide, South Australia, Australia
  15. 15Bioinformatics Core Facility, Swiss Institute of Bioinformatics, Lausanne, Switzerland
  16. 16Department of Medicine, Columbia University Medical Center, New York City, New York, USA
  17. 17Digestive Oncology Unit, Department of Oncology, University Hospitals Leuven, Leuven, Belgium
  18. 18Royal Brisbane and Womens Hospital, Brisbane, Queensland, Australia
  19. 19School of Medicine, University of Queensland, Brisbane, Queensland, Australia
  20. 20Pathology Queensland, Brisbane, Queensland, Australia
  1. Correspondence to Dr Daniel L Worthley, South Australian Health & Medical Research Institute, Adelaide SA, Australia; Dan.Worthley{at}sahmri.com and Dr Susan L Woods, School of Medicine, University of Adelaide and South Australian Health and Medical Research Institute, Adelaide, SA 5005, Australia; susan.woods{at}adelaide.edu.au

Abstract

Objective Serrated colorectal cancer (CRC) accounts for approximately 25% of cases and includes tumours that are among the most treatment resistant and with worst outcomes. This CRC subtype is associated with activating mutations in the mitogen-activated kinase pathway gene, BRAF, and epigenetic modifications termed the CpG Island Methylator Phenotype, leading to epigenetic silencing of key tumour suppressor genes. It is still not clear which (epi-)genetic changes are most important in neoplastic progression and we begin to address this knowledge gap herein.

Design We use organoid culture combined with CRISPR/Cas9 genome engineering to sequentially introduce genetic alterations associated with serrated CRC and which regulate the stem cell niche, senescence and DNA mismatch repair.

Results Targeted biallelic gene alterations were verified by DNA sequencing. Organoid growth in the absence of niche factors was assessed, as well as analysis of downstream molecular pathway activity. Orthotopic engraftment of complex organoid lines, but not BrafV600E alone, quickly generated adenocarcinoma in vivo with serrated features consistent with human disease. Loss of the essential DNA mismatch repair enzyme, Mlh1, led to microsatellite instability. Sphingolipid metabolism genes are differentially regulated in both our mouse models of serrated CRC and human CRC, with key members of this pathway having prognostic significance in the human setting.

Conclusion We generate rapid, complex models of serrated CRC to determine the contribution of specific genetic alterations to carcinogenesis. Analysis of our models alongside patient data has led to the identification of a potential susceptibility for this tumour type.

  • colorectal cancer
  • gene mutation
  • cancer genetics
  • oncogenes
  • methylation

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Footnotes

  • DLW and SLW contributed equally.

  • Contributors DLW and SLW devised the study and wrote the manuscript. YH, SJL, HEA, OHY, JM, SM, ST, BAL and VLJW provided intellectual input into study design. TRML, YKL, LV, RS, MY, JAW and SLW generated and analysed organoid lines including CRISPR/Cas9 gene editing, DNA sequencing, qPCR and western blot. TRML, TW, JR, KG, JQN, NS, MI and TLP carried out animal experiments. TW, PW and SLW analysed the RNAseq and TCGA data. LF and VLJW performed and analysed MSI. TRML, MLB, LJ, SK and HK performed and analysed (histo)pathology. All authors read and approved the final manuscript.

  • Funding This work was supported by Cure Cancer Australia/Cancer Australia (APP1102534), the Cancer Council SA Beat Cancer Project on behalf of its donors and the State Government of South Australia through the Department of Health SA, by Pathology Queensland, by the Australian National Health and Medical Research Council (NHMRC) (through APP1081852, APP1140236), by the National Institutes of Health (NIH) (K08 CA198002, R00 AG045144, R01 CA211184), the Department of Defense (CA120198), the V Foundation V Scholar Award, the Sidney Kimmel Scholar Award, the Pew-Stewart Trust Scholar Award, the Koch Institute Frontier Research Program through the Kathy and Curt Marble Cancer Research Fund, the American Federation of Aging Research, as well as by the Koch Institute Support Grant P30-CA14051 from the National Cancer Institute. DLW is supported by a NHMRC Career Development Fellowship, VLJW by a Gastroenterological Society of Australia Senior Research Fellowship.

  • Competing interests None declared.

  • Patient consent Not required.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data sharing statement mRNAseq data are available through the NCBI GEO website under accession GSE112774.

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